PROJECT SUMMARY Human mesenchymal stem cells (hMSCs) are under intense research for applications in cell and gene therapeutics due to ease of isolation from multiple adult tissues and ability to home to sites of injury and reduce inflammation. hMSCs are also immune evasive, allowing for allogeneic transplantation for off-the-shelf therapies. While their unique properties give them great potential in therapeutic applications, hMSCs intrinsic therapeutic properties could be greatly enhanced by gene delivery. Gene delivery through viral transduction is efficient, but suffers from safety issues related to immunogenicity and insertional mutagenesis, however, non-viral gene delivery, while safer compared to viral, suffers from inefficiency and low transgene production, especially in hMSCs. To address the shortcomings of non-viral gene delivery to hMSCs, our group has demonstrated that pharmacological ?priming?, such as with the glucocorticoid dexamethasone (DEX), can significantly increase non- viral gene delivery to hMSCs by modulating transfection-induced cytotoxicity. As a part of the Parent Award, we have expanded our hMSC priming library by screening 707 FDA approved drugs and identified new candidate drugs that can significantly increase transgene production and transfection efficiency compared to a vehicle control in two different donors of adipose-derived mesenchymal stem cells. Here, we now propose to perform systematic comparison of key attributes of the non-viral gene delivery system (i.e. DNA vector, promoter, number of transgenes, expression controllers), in combination with drugs from our newly expanded priming library, to develop an efficient strategy to deliver multiple genes into mesenchymal stem cells, so that these cells could be advanced as a cell therapy for Alzheimer?s disease (AD). To address AD pathogenic features, current research and clinical trials have focused on disease-modifying monotherapies, such as anti-aggregation drugs to inhibit amyloid plaques and tau tangles, delivery of neurotrophic factors to promote neurogenesis, and stem cell- and gene-based therapies to reduce inflammation and neurodegeneration. While these monotherapies successfully modified the intended target, all failed to halt AD progression and reverse cognitive decline, suggesting that each hallmark of AD in and of itself is not the main driver of AD progression, but that a complex interaction between each pathogenic feature may be driving AD. Therefore, this proposal seeks to develop an efficient non-viral gene delivery system that makes use of priming for genetic modification of adipose-derived hMCSs (AMSCs) to express three genes: membrane metallo-endopeptidase (MME) (i.e. neprilysin (NEP), an A? degrading enzyme), protein phosphatase 2 regulatory subunit Balpha (PP2A/B?) (a tau targeted phosphatase), and glucagon-like peptide-1 (GLP-1) (a neurotrophic signaling enhancer) for degradation of amyloid plaques, regulation of tau phosphorylation, and reversal of neurodegeneration, respectively, through two aims: (1) Screen DNA vectors, promoters, number of genes incorporated, and priming library for efficient delivery of exogenous DNA to AMSCs. (2) Evaluate efficacy of non-virally modified AMSCs in ameliorating AD hallmarks in vitro.